Our understanding of the mechanisms mediating resistance to myocardial ischemia remains unclear. To determine mechanisms for increasing resistance to ischemia, we subjected hearts from Brown Norway (BN/Mcw) and Dahl S (SS/Mcw) rats to global ischemia. Hearts from BN/Mcw rats were more resistant to ischemia than hearts from SS/Mcw rats. Examination of cellular mechanisms revealed that although hearts from both strains exhibited the same levels of endothelial nitric oxide synthase (NOS3) and heat shock protein 90 (hsp90) expression, hearts from BN/Mcw rats generated more nitric oxide (.NO) and less superoxide anion (02.-) than hearts from SS/Mcw rats. Basic proteomic studies revealed that the shift in the balance in NO and O2.- production toward NO in the BN/Mcw hearts was due, at least in part, to altered heat shock protein 90 (hsp90) association with NOS3 and possibly, with GTP cyclohydrolase I (GTPCH-I). We observed that hsp90 association with NOS3 in BN/Mcw hearts was increased nearly 2-fold compared to association in SS/Mcw hearts; and that total biopterin, an analytical index of tetrahydrobiopterin (BH4) was increased in BN/Mcw hearts by 80%. Total biopterin concentrations in hearts from BN/Mcw rats directly correlated with a 70% increase in GTPCH-I protein levels and interestingly, a 2.2-fold increase in association of GTPCH-I with hsp90 compared to the levels in SS/Mcw hearts. These data suggest that hsp90-dependent chaperone activity may play a critical role in mechanisms governing NOS3 function that many consider to be two independent hypotheses: 1) hsp90-dependent signaling modulates NOS3 generation of .NO (coupled activity) and O2.- (uncoupled activity); and, 2) modulation of BH4 levels, which in turn, regulate coupled and uncoupled NOS3 activity. The overall objective of this proposal is to determine the cellular, molecular and enzymatic mechanisms by which hearts from BN/Mcw rats are more resistant to ischemia than hearts from SS/Mcw rats. By elucidating these pathways, findings from this proposal will provide new, fundamental insight into how hsp90 chaperone activity increases NOS3 .NO generation to increase resistance to ischemia. Observations from these studies may actually unify two hypotheses that many consider competing hypotheses in cardiovascular physiology and may also lead to the development of new strategies for treating ischemic heart disease.